Metastatic disease is the major cause of death from cancer. However, just as not all primary cancers are prone to metastasize, not all tumor cells found at secondary sites are life-threatening. Dissemination from a primary tumor site can occur relatively early in tumor development, and cells at secondary sites may have properties that range from indolence to aggressive malignancy. This proposal is designed to address the provocative question How do we determine the significance of finding cells from a primary tumor at another site and what methods can be developed to make this diagnosis clinically useful? successfully answering this question would be expected to identify new Achilles' heels for tumors which could be the basis for developing novel therapeutics. The rationale for the proposed research is based on our preliminary studies showing that human breast cancer xenografts disseminate cells around the body and that these cells have distinct profiles that differ from the parent tumor. Given our novel insights, we now propose to analyze primary tumors and disseminated cells from human xenograft models of breast cancer for biomarkers of dissemination and metastasis; determine the heterogeneity of the cells in these populations by characterizing gene expression in single cells using micro-fluidic single cell PCR analysis; determine whether the disseminated tumor cells have the ability to initiate tumors upon transplantation; analyze the characteristics of the microenvironment of the disseminated tumor cells in which they flourish or remain indolent; and validate the hits for dissemination and metastasis. These studies will open up further studies with transplant of single cells of specific phenotypes, to learn which changes are crucial for metastasis. Understanding the molecular mechanisms of this complex interplay between malignant cancer cells and their surrounding non-malignant stroma represents one of the major challenges in cancer research, which once understood, will foster a better understanding of which disseminated tumor cells are quiescent and which are likely to give rise to metastases aggressively. This understanding will form the basis for new biomarkers and pharmacological interventions.